Efficient algorithm for PCR testing of blood samples

ABSTRACT

Systems, processes, and devices are provided which are useful for testing blood or plasma donations to detect those specific donations which are contaminated by a virus above a predetermined level. An apparatus and process is described which forms individual, separately sealed and connected sample containers from a flexible hollow tubing segment connected to a fluid donation container. The tubing segment is sealed at spaced-apart intervals along its length, with tubing segment portions in the intervals between the seals defining containers, each of which holds a portion of a plasma sample. The contents of the containers are formed into pools which are subsequently tested for virus contamination by a high-sensitivity test such as PCR. The pools are tested in accordance with an algorithm by which a sample from each donation is mapped to each element of an N-dimensional matrix or grid. Each element of the matrix is identified by a matrix identifier, X rcs , where rcs defines the dimensional index. An aliquot is taken from each sample, and subpools are formed, each subpool comprising aliquots of samples in which one dimensional index is fixed. All of the subpools are tested in one PCR test cycle. The dimensional indicia of each positive subpool is evaluated mathematically in accordance with a reduction by the method of minors, thereby unambiguously identifying a unique element in the grid, thereby unambiguously identifying a uniquely positive blood or plasma donation.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a division of patent application No.09/549,477, filed Apr. 14, 2000, now U.S. Pat. No. 6,566,052, which is adivision of patent application No. 09/081,926, filed May 20, 1998, nowU.S. Pat. No. 6,063,563, which is a division of patent application No.08/778,610, filed Jan. 6, 1997, now U.S. Pat. No. 5,780,222; which is acontinuation-in-part of patent application No. 08/683,784, filed Jul.16, 1996, now U.S. Pat. No. 5,834,660; which is a division of patentapplication No. 08/419,620, filed Apr. 10, 1995, now U.S. Pat. No.5,591,573.

FIELD OF THE INVENTION

[0002] The present invention relates generally to systems and processesfor preparing and analyzing samples taken from plasma donations touniquely identify donations which are virus contaminated. In particular,the invention relates to an apparatus and process for formingindividual, separately sealed, and connected containers holding samplesof the same plasma as is contained in the donation. The invention alsorelates to an apparatus and process for forming initial screening testpools from the containers and testing the pools for the presence of avirus in accordance with an algorithm to identify individualcontaminated donations in the fewest number of testing cycles.

BACKGROUND OF THE INVENTION

[0003] Blood, plasma, and biological fluid donation programs areessential first steps in the manufacture of pharmaceutical and bloodproducts that improve the quality of life and that are used to savelives in a variety of traumatic situations. Such products are used forthe treatment of immunologic disorders, for the treatment of hemophilia,and are also used in maintaining and restoring blood volume in surgicalprocedures and other treatment protocols. The therapeutic uses of blood,plasma, and biological fluids require that donations of these materialsbe as free as possible from viral contamination. Typically, a serologytest sample from each individual blood, plasma, or other fluid donationis tested for various antibodies, which are elicited in response tospecific viruses, such as hepatitis C (HCV) and two forms of the humanimmunodeficiency virus (HIV-1 and HIV-2). In addition, the serology testsample may be tested for antigens designated for specific viruses suchas hepatitis B (HBV), as well as antibodies elicited in response to suchviruses. If the sample is serology positive for the presence of eitherspecific antibodies or antigens, the donation is excluded from furtheruse.

[0004] Whereas an antigen test for certain viruses, such as hepatitis B,is thought to be closely correlated with infectivity, antibody tests arenot. It has long been known that a blood plasma donor may, in fact, beinfected with a virus while testing serology negative for antibodiesrelated to that virus. For example, a window exists between the timethat a donor may become infected with a virus and the appearance ofantibodies, elicited in response to that virus, in the donor's system.The time period between the first occurrence of a virus in the blood andthe presence of detectable antibodies elicited in response to that virusis known as the “window period.” In the case of HIV, the average windowperiod is approximately 22 days, while for HCV, the average windowperiod has been estimated at approximately 98 days. Therefore, testsdirected to the detection of antibodies, may give a false indication foran infected donor if performed during the window period, i.e., theperiod between viral infection and the production of antibodies.Moreover, even though conventional testing for HBV includes tests forboth antibodies and antigens, testing by more sensitive methods haveconfirmed the presence of the HBV virus in samples which were negativein the HBV antigen test.

[0005] One method of testing donations, which have passed availableantibody and antigen tests, in order to further ensure their freedomfrom incipient viral contamination, involves testing the donations by apolymerase chain reaction (PCR) method. PCR is a highly sensitive methodfor detecting the presence of specific DNA or RNA sequences related to avirus of interest in a biological material by amplifying the viralgenome. Because the PCR test is directed to detecting the presence of anessential component of the virus itself, its presence in a donor may befound almost immediately after infection. There is, theoreticallytherefore, no window period during which a test may give a falseindication of freedom of infectivity. A suitable description of themethodology and practical application of PCR testing is contained inU.S. Pat. No. 5,176,995, the disclosure of which is expresslyincorporated herein by reference.

[0006] PCR testing is, however, very expensive and since the generaldonor population includes a relatively small number of PCR positivedonors, individual testing of each donation is not cost effective oreconomically feasible. Hence, an efficient and cost-effective method oftesting large numbers of blood or plasma donations to eliminate unitshaving a viral contamination above a predetermined level is required.

[0007] One method of testing a large number of plasma donations is topool a number of individual plasma donations. The pool is then PCRtested and the individual donations comprising the pool are eitherretained or disposed of, depending on the outcome of the PCR test. Whilereducing the number of PCR tests, and the costs associated therewith,this method results in a substantial waste of a significant portion ofvirus free donations. Since only a single donation with a viralcontamination above a pre-determined level will cause a pool to test PCRpositive, the remaining donations that contribute to a pool may well beindividually PCR negative. This result is highly probable given that arelatively small number of PCR positive donors exist in the generaldonor population. In the conventional pooling approach, all donationscomprising the pool are disposed of upon a PCR positive result,including those donations that are individually PCR negative.

[0008] In addition, plasma donations are often frozen soon after theyare received. When samples of individual plasma donations are needed forpooling, each donation must be thawed, an aliquot of the blood or plasmaremoved from the donation, and the donation must then be refrozen forpreservation. Multiple freeze-thaw cycles may adversely affect therecovery of the RNA or DNA of interest as well as the proteins containedwithin the plasma, thus adversely affecting the integrity of the PCRtest. Moreover, each time an aliquot of individual plasma donations iswithdrawn to form a pool, the donation is subject to contamination, bothfrom the surrounding environment, and from the apparatus used towithdraw the aliquot. Further, if the donation contains a virus, it cancontaminate other donations. In order to avoid introducing viralcontaminants into an otherwise viral free donation, the sample takingapparatus must be either sterilized after each individual use, or usedfor taking only a single aliquot from a single individual donation and anew sample taking apparatus used for taking an aliquot from a subsequentindividual donation. Either of these methods involves considerableexpense and is quite time consuming.

[0009] Accordingly, there is a need for a process and system forobtaining multiple blood or plasma samples from individual donationssuch that particular samples may be pooled without contaminating theremaining samples. It is also desirable that the process and system isable to form such pools in a fast and efficient manner, withoutcontaminating either a clinical testing lab technician or the testinglaboratory environment.

[0010] In addition, it is desirable that the process and system providefor efficient and cost-effective testing of the blood or plasmadonations to identify only uniquely PCR positive donations in the fewestpossible number of testing cycles.

SUMMARY OF THE INVENTION

[0011] There is, therefore, provided in the practice of this invention acost-effective and efficient process for preparing and testing samplesfrom a multiplicity of blood or plasma donations to uniquely identifydonations which are infected with virus as well as systems and devicesfor practicing the process.

[0012] The process of the present invention results in blood and plasmaproducts being substantially safer because one can readily test forvirus contamination in the blood or plasma supply directly.Cost-effective, high-sensitivity testing can be performed immediately,and contaminated donations identified, without regard to an infectivitywindow period.

[0013] In one embodiment of practice of the present invention, theprocess comprises the steps of providing a blood or plasma donation in acollection container. A flexible collection segment is connected to thecontainer and is open to the inside of the container. The collectionsegment is filled with blood or plasma from the collection container,and a portion of the collection segment is sealed at both ends. Thesealed portion of the collection segment is removed from the containerand, either before or after the sealed collection segment portion isremoved, a plurality of spaced-apart seals are provided at intervalsalong the length of the collection segment between the sealed ends. Thesegment portions in the intervals between adjacent seals definecontainers, wherein each such container contains a plasma or bloodsample, and wherein the intervals between the seals provide a sufficientvolume in each such container for the planned testing.

[0014] In a more detailed embodiment of the present invention,individual plasma donations are collected in a plasma collection bottlewhich has a testing container connected thereto by a flexible hollowtubing segment. After being filled with a donor's plasma the plasmabottle is tipped so as to transfer plasma to the testing container andthe flexible tubing segment, thereby filling the tubing segment. Thetubing segment is sealed at spaced-apart intervals along its length, thetubing segment portions in the intervals between the seals definepouches each of which contains a sample of the plasma donation. Thetubing segment, which has been converted into a series of pouches, isthen disconnected from the plasma collection bottle and frozen untilneeded for testing.

[0015] In an additional aspect of the present invention, the hollowtubing segment comprises a series of linked-together Y-sites, includingan injection site provided on one leg of the Y, and where each branchleg of a particular Y-site which does not include an injection site isconnected to the base of the next Y-site in the chain by a flexibleplastic tubing segment. Spaced-apart heat seals are formed along thelength of each flexible plastic tubing segment separating the Y-sites.

[0016] In a further aspect of the present invention a device forproviding multiple heat seals along the length of the tubing segmentfilled with the blood or plasma donation comprises first and secondopposed seal platens. Each seal platen includes a plurality ofspaced-apart raised portions along its length alternating with recessedportions. The raised and recessed portions on the first platen are inregistry with corresponding raised and recessed portions on the secondplaten. The opposed seal platens are moved together onto a plastictubing segment filled with the blood or plasma donation to form heatseals on those portions of the tubing segment compressed between theraised portions and to form chambers defined by opposed recessedportions. The heat seals define a plurality of individual and sequentialpouches therebetween and each chamber, defined by each closed pair ofrecessed portions, is configured to house a pouch.

[0017] In particular, a device for providing multiple heat seals alongthe length of the tubing segment filled with a blood or plasma donationis configured to be mounted on a commercially available heat sealapparatus, as an after-market modification.

[0018] In yet a further embodiment of the invention, a system forcollecting and preparing plasma samples for testing comprises a plasmacollection container and a hollow plastic tube connected to thecontainer, each of which are constructed of plastic and each of whichcontain a coded indicia molded into the plastic. The coded indicia isdisposed along the major axis of the tubing segment and the code repeatsat spaced-apart intervals so that the tubing segment can be providedwith a plurality of spaced apart seals along its length to therebydefine pouches between the seals. The code intervals of the indiciacorrespond to the intervals of the pouches, so that each pouch willcontain at least one cycle of the code.

[0019] To begin the testing process of the present invention, a firstpouch is removed from each of a group of tubing segments correspondingto a plurality of separate plasma donations. A portion of the contentsof each such first pouch is withdrawn and the contents formed into apool in a container.

[0020] In an exemplary embodiment of the present invention, the firstpool is tested for a viral indication. When the first pool testspositive for a viral indication, a next, or second, sequential pouch isremoved from each of the tubing segments that were used to form thefirst pool. The second pouches are divided into two approximately equalsubgroups, and the contents of one of the subgroup pools is tested forthe presence of a specific virus. When the tested subgroup pool testsnegative for the virus, a further sequential pouch is removed fromcorresponding tubing segments used to form the untested subgroup. Thepouches are divided into two approximately equal next generationsubgroups, and the contents of the subgroup pouches are formed intopools. One of the next generation subgroup pools is tested for a viralindication.

[0021] When the tested subgroup pool tests positive for such viralindication, a pouch is removed from corresponding tubing segments usedto form the tested subgroup. The process is iterated, with each positivepool being further subdivided into successively smaller subgroups, witheach of the successive subgroups comprising a fraction of the samples ofthe preceding positive subgroup, until the final pouch corresponding toa single plasma donation is identified.

[0022] In a further embodiment of the present invention, an additionalprocess for testing a multiplicity of plasma donations to uniquelyidentify donations having a positive viral indication in a single PCRtesting cycle includes the steps of defining an n-dimensional grid whichdefines internal elements at the intersections of each of then-dimensions of the grid. A sample from each of a number of plasmadonations is mapped to a corresponding element of the grid, with eachsample being defined by a matrix notation, X_(rcs), where the subscriptof the matrix element notation defines dimensional indices of the grid.Aliquots are taken from each sample of each of the plasma donations andformed into subpools. Each subpool includes an aliquot of all plasmadonation samples in which one of the dimensional indices is fixed. Thesubpools are all tested at once, in a single PCR testing cycle, and thedimensional indicia of each subpool which tests positive is evaluated inaccordance with a reduction by the method of minors, therebyunambiguously identifying a unique element defined by the dimensionalindicia of each positive subpool, and thus unambiguously identifying auniquely positive sample.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] These and other features, aspects, and advantages of the presentinvention will be more fully understood when considered with respect tothe following detailed description, appended claims, and accompanyingdrawings, wherein:

[0024]FIG. 1 is a semi-schematic perspective view of one example of aplasma donation bottle and sample container attached by a tubing segmentuseful in the practice of the present invention;

[0025]FIG. 2 is a semi-schematic perspective view of a tubing segmentconnected between a plasma donation bottle and sample container anddivided into pouches in accordance with the present invention;

[0026]FIG. 2a is a semi-schematic perspective view of a tubing segmentconnected between a plasma donation bottle and sample container andincluding a series of linked-together Y-sites in accordance with thepresent invention;

[0027]FIG. 3a is an enlarged top plan view of a portion of the tubingsegment shown in FIG. 2 showing additional details of the seals whichseparate the pouches;

[0028]FIG. 3b is a semi-schematic cross-sectional view of a tubingsegment seal;

[0029]FIG. 4 is a semi-schematic perspective view of a device providedin accordance with practice of the present invention for sealing atubing into individual pouches;

[0030]FIG. 4a is a semi-schematic perspective view of a top and bottomplatens of a heat sealing device provided in accordance with practice ofthe present invention for mounting onto a commercially available heatsealer;

[0031]FIG. 5 is a semi-schematic perspective view of a sampling plateand cover provided in accordance with the present invention;

[0032]FIG. 6 is a semi-schematic partial cross-sectional view of aplasma pouch contained in a sampling plate sample well provided inaccordance with the present invention;

[0033]FIG. 7 is a semi-schematic perspective view of a device providedin accordance with the present invention for crushing sample pouches andexpressing the fluid samples container therein into a pool;

[0034]FIG. 8 is a semi-schematic cross-sectional view of a crushingcylinder of the device of FIG. 7;

[0035]FIG. 9a is a semi-schematic partial cross-sectional view of ascreen plate against which sample-containing packets are crushed;

[0036]FIG. 9b is a semi-schematic top view of a screen plate showingradial and concentric fluid gutters for collecting sample fluid fromcrushed sample containers;

[0037]FIG. 10 is a semi-schematic partial cross-sectional view of acrushing piston of the device of FIG. 7;

[0038]FIG. 11 is a flow chart depicting the test methodology accordingto the invention for determining PCR positive donors from a donationpool;

[0039]FIG. 12 is a flow chart depicting a test sequence according to theinvention for identifying a single PCR positive donation from a 512donation pool;

[0040]FIG. 13 is a flow chart depicting a second test methodologyaccording to the invention for determining PCR positive donors from adonation pool; and

[0041]FIG. 14 is a representation of a 3-dimensional grid according tothe invention showing the definition of r, c, and s indices.

DETAILED DESCRIPTION

[0042] The present invention relates to systems, processes and devicesuseful for testing blood or plasma donations to detect those specificdonations which have a viral contamination above a predetermined level.Such contaminated donations are then disposed of to thereby preventtheir incorporation into the raw material stream for pharmaceuticalproducts or their transfusion into human patients. The viral detectiontests used in accordance with practice of the present invention can beany that directly detect a virus instead of antibodies elicited inresponse to the virus. The tests include polymerase chain reaction (PCR)tests and other tests which are sufficiently sensitive to directlydetect a virus even after pooling samples from multiple donations.

[0043] In one embodiment of practice of the present invention, aplurality of separate blood or plasma donations are provided. A blood orplasma sample is drawn from each donation into a corresponding flexible,hollow tubing segment. A plurality of spaced-apart seals are provided atintervals along the length of the tubing segment, so that segmentportions in the intervals between seals define pouches where each pouchcontains a blood or plasma sample. As is discussed below in greaterdetail, a unique methodology is provided in accordance with the presentinvention for testing plasma samples from the pouches after the samplesare formed into pools to thereby efficiently and effectively detect andisolate any such blood or plasma donation which is contaminated withvirus.

[0044] Turning to FIG. 1, an exemplary embodiment of a system providedin accordance with practice of the present invention for effecting thesampling process is shown. The system includes a standard plasmadonation container 20, constructed of a nonreactive material such aspolyvinyl chloride (PVC). The donation container 20 includes a cap 22having two hollow elbow shaped fittings 23 and 24, respectively,attached to the top surface thereof. The fittings communicate with theinterior of the donation bottle through orifices provided in cap 22 forsuch purpose. A flexible hollow filler tube 26, constructed of abiologically neutral material, such as PVC plastic, is connected at oneend to the elbow fitting 23 and connected at the other end to, forexample, a needle which is inserted into a donor in order to procure adonation. In the illustrated embodiment, a test container 28, is alsoprovided, for collecting a sample from the donation to be serologytested. The test container 28 is generally test tube shaped and is alsoconstructed of a biologically nonreactive material. The test container28 includes an integral cap member 30 through which orifices areprovided in order to communicate with the interior of the testcontainer.

[0045] A flexible hollow tubing segment 32, constructed of abiologically nonreactive plastic material, is connected between the capmember 30 of the test container 28 and the hollow elbow fitting 24 ofthe plasma donation container cap. The tubing segment 32 is connected tothe cap member 30 in a manner such that fluid passing through the tubingsegment will enter the test container 28 through an orifice provided inthe cap member 30 for such purpose. The tubing segment 32 may befriction fit into said orifice, sonically welded thereto, or otherwiseattached in a coaxial relationship with the orifice by techniques wellunderstood by those skilled in the art.

[0046] A second orifice may also be provided in the cap member 30, towhich a vent tube 34 is connected in a manner similar to tubing segment32. The vent tube 34 is typically no more than one to two inches inlength, and is typically terminated with an inserted, friction fitbacteria excluding filter 36.

[0047] In an exemplary embodiment, a blood or plasma donation iswithdrawn from a donor and collected in the plasma donation container 20for subsequent storage until needed. In the case of a plasma donation,blood is typically withdrawn from a donor and passed through acontinuous centrifuge apparatus, wherein red blood cells are centrifugedout from the supporting plasma fluid and returned to the donor. Theplasma is then collected.

[0048] After a plasma donation is taken from a donor and the donationcontainer 20 is filled, the donation container is tilted so as to raisethe fluid level over the elbow fitting 24 connected to the tubingsegment 32. Plasma enters the tubing segment, flows through the tubingsegment, and fills the test container 28. During filling, air trappedwithin test container 28 escapes through the vent tube 34, allowing thetest container to be filled completely. The bacteria excluding filter 36filters out any bacteria in the returning air, thus preventingcontamination of the sample by the surrounding environment. After thetest container is filled, plasma from the donation is allowed to fillthe tubing segment 32.

[0049] Turning now to FIG. 2, after the plasma sample from the donationis drawn into the tubing segment 32, the tubing segment is sealed by aheat weld 38 or other suitable sealing means such as a sonic weld, at alocation proximate to the tubing segment's connection to the plasmadonation container. A further heat seal 40 is applied to the tubingsegment at a location proximate to the segment's connection to the testcontainer 28. An elongated hollow tube, closed off at both ends, andcontaining a quantity of the plasma donation is thus provided.

[0050] The filled portion of the tubing segment 32 is removed from theplasma donation and test containers by cutting the tubing segment awaythrough the center of the seals, 38 and 40. The separate plasma donationcontainer is then removed for freezing and storage, while the separatedtest container is removed to a laboratory for serology testing.Typically, the contents are tested for various antibodies, which areelicited in response to specific viruses, such as hepatitis C (HCV), orHIV-1 and HIV-2.

[0051] Additional seals 42 are also provided at spaced-apart intervalsalong the length of the tubing segment, to define sequential individualand connected pouches, each suitably comprising a hollow tubing segmentportion 44. Each such portion 44 contains a particular quantity of bloodor plasma needed for the specific generation pool to be formed. Forexample, for pouches to be formed for PCR testing, approximately 0.02 to0.5 ml of blood or plasma from the host donation may be sealed.

[0052] The tubing segment is sealed in a manner to provide from 5 to 15,individual and connected pouches. Sealing, to define the pouches, may bedone either after the tubing segment has been removed from between theplasma donation container and the serology test container or preferablyis done while the tubing segment is still attached to the plasmadonation container, in order to avoid hydrostatic pressure build-up.Sealing may be done by any known method, such as thermo-compressionsealing (heat sealing), sonic welding or the like, so long as the lengthof the region compressed and sealed is sufficient to permit theconnected pouches to be separated from one another by cutting throughthe center of the seal without violating the integrity of the pouch oneither side, as indicated more clearly in FIGS. 3a and 3 b. A secondembodiment of the tubing segment adapted to be subdivided into blood orplasma sample-containing aliquot portions is depicted in FIG. 2a whichis a semi-schematic perspective view of a collection tubing segmentembodiment connected between a plasma donation bottle 20 and samplecontainer 28 and divided into aliquot-containing portions in accordancewith the present invention.

[0053] The collection tubing segment 50 is connected between the capmember 30 of the test container 28 and the hollow elbow fitting 24 ofthe plasma donation container cap. The tubing segment 50 suitablycomprises a plurality of Y-sites 51 connected together in series byflexible, hollow, medical-grade plastic tubing segments 52. The Y-sites51 are of the type commonly adapted for connection to an intravenousinfusion set and include a cylindrical body portion 53 with a flow pathdefined therethrough, having an outlet 54 at one end of the flow pathand an access site 55 at the other end. A branch port 56 is providedalong the body 53 of the Y-site and includes a fluid path which is incommunication with the fluid path through the body 53.

[0054] One Y-site is connected to the next by solvent bonding aflexible, hollow, medical-grade plastic tube 52 between the outlet port54 on the bottom of one Y-site to the branch port 56 of the next Y-sitein the series. An initial hollow entry tube 57 is solvent bonded to thebranch port of the initial Y-site in the series. The initial entry tube57 is connected, in turn, to the elbow fitting 24 of the plasma donationcontainer cap. Connection may be made by friction-fitting the initialentry tube 57 onto the elbow fitting 24, sonically welding the tubethereto, or otherwise attaching the tube in a coaxial relationship withthe fitting by techniques well understood by those skilled in the art.Moreover, the initial entry tube 57 may terminate in a standardluer-type fitting 58 which would allow the series-connected Y-sites tobe removably connected to a donation container which was provided with amating luer-type connector at the end of the elbow 24.

[0055] In like manner, the terminal Y-site is fitted with a flexible,hollow terminal exit tube 59 which is solvent-bonded to the terminalY-site at its outlet port. This tube may also be connected to a standardluer-type fitting at its distal end.

[0056] In a manner similar to that described in connection with thefirst embodiment, after a plasma donation is taken from a donor and thedonation container 20 is filled, the donation container is tilted so asto raise the fluid level over the elbow fitting 24 connected to theentry tube segment 57. Plasma enters the tubing segment and flowsthrough the series-connected Y-sites, entering each Y-site through itsbranch port 56 and flowing into the next Y-site from the precedingY-site's outlet port 54. Plasma is decanted until the test container 28is filled. After the test container is filled, the donation is furtherdecanted until the series-connected Y-sites comprising the tubingsegment 50 are also filled.

[0057] After the plasma sample from the donation is drawn into thetubing segment 50, the terminal exit tubing segment 59 is closed off bya heat seal or weld 40 a or other suitable sealing means such as a sonicweld at a suitable location along its length proximate to the terminalexit tubing segment's connection to the test container 28.

[0058] The filled tubing segment 50 is removed from the test containerby cutting the terminal exit tubing segment 59 away from the testcontainer through the center of the seal 40 a. Alternatively, if thetubing segment 50 terminates in a luer-type connector, the tubingsegment 50 is removed from the test container 28 by disconnecting theluer. A second heat seal 38 a is applied to the initial entry tubingsegment 57 at a location along its length proximate to the initialsegment's connection to the donation container 20. The filled portion ofthe tubing segment 50 is removed from the plasma donation by cutting theinitial entry segment 57 away through the center of the seal 38 a, or bydisconnecting the luer-type fitting 58, if such is provided. Anelongated, hollow, articulated tube, closed off at both ends andcomprising a plurality of Y-sites linked-together in series, is thusprovided. Each of the linked-together Y-sites contains an aliquot of theblood or plasma donation.

[0059] As will be described in greater detail below, the tubing segmentsconnecting a preceding Y-site's outlet port to a subsequent Y-site'sbranch port are also provided with heat seals 42 a to define sequential,individual, and connected sample aliquots, each suitably comprising anindividual Y-site. Each such Y-site contains a particular quantity ofblood or plasma needed for a specific generation pool to be formed.Sealing to isolate each Y-site may be performed either after the tubingsegment 50 has been removed from the plasma donation container or may beperformed while the tubing segment is still attached. Preferably,sealing to isolate the Y-sites is performed while the tubing segment 50still attached to the plasma donation container so that the volumereduction caused flattening a portion of the tubing during the sealingprocess does not cause a build-up in the internal hydrostatic pressureof the sample. When the tubing segment 50 remains connected to theplasma donation container, excess fluid created by the volume reductionof the tubing created by the heat seals is allowed to be expressed backinto the donation container. Excess hydrostatic pressure, which may leadto dangerous spurting during sample extraction, is thus safely relieved.

[0060] Sealing may be performed by any known method, such asthermo-compression sealing (heat sealing), sonic welding or the like, solong as the length of the region which is compressed and sealed issufficient to permit the connected Y-sites to be separated from oneanother by cutting through the center of the seal without violating theintegrity of the tubing segment on either side of the seal.

[0061] Turning now to FIGS. 3a and 3 b, in a preferred embodiment, theseal between pouches (42 of FIG. 2) and/or Y-sites (51 of FIG. 2a)includes a flat pad area 46, including a central narrow portion 47through which the seal is cut or torn in order to separate the connectedpouches. Cutting is done through the central portion in order to insurethat each separated pouch remains sealed at compressed tab portions 48at either end after separation. The length of the seal pad may be madegreater or smaller, depending on the chosen separation method.Separation may be done by use of a scalpel, a guillotine cutter, or asimple pair of scissors.

[0062] Turning to FIG. 4, an exemplary embodiment of a sealing device60, useful for providing pouches of specific desired sizes, includingmeans to easily separate the pouches and identify their sequence numberalong a segment, is shown. The sealing device 60 suitably comprisesopposed first and second platens 61 and 62, respectively, each includinga plurality of raised, seal head portions 63, arranged in a spaced apartrelationship on the opposing surfaces of the platen. The sealing device60 is preferably constructed such that the raised seal head portions 63are movable along their respective platens such that the spacing fromone raised seal head portion to another may be varied. The raised sealheads 63 may be arranged along the platen such that the distance betweensuccessive seal heads is made progressively smaller so that sealing isperformed along the length of a tubing segment at progressively closerspaced intervals. Thus, sample pouches of progressively smaller sizeand, therefore, progressively smaller volume content may be formed bymoving pairs of opposed seal heads along their respective platens to adesired location.

[0063] In order to form multiple heat seals along the length of theplastic tubing segment filled with a blood or plasma sample, the tubingsegment is placed within the sealing device 60 between the upper andlower sealing platens 61 and 62, respectively. The opposed platens arebrought into proximity with one another, thus compressing and sealingthe tubing segments. As depicted in FIG. 4, the plurality ofspaced-apart, extended or raised seal head portions 63 along the lengthof each platen alternate with recessed portions 64. As the opposedplatens are moved together to form heat seals on those portions of aplastic tubing segment filled with a blood or plasma sample compressedbetween the raised seal head portions 63, chambers are formed by theopposed recessed portions 64. The chambers are provided in order toaccommodate those portions of the tubing segment which are not to becompressed but, rather, to be formed into pouches. Each chamber definedby each closed pair of recessed portions is configured to house a pouch.

[0064] A heater 65 is configured to heat each of the seal head portionsof the platen in order for opposed raised portions to form a heat sealon the tubing segment when the sealing device is closed. The heater 65may be any one of well known heater types such as radiant heaters,induction or resistance heaters, or the like. The heater 65 ispreferably connected directly to each of the raised seal heads 63 toheat the raised portions without unduly heating the recesses. Ifdesired, insulation can be provided to reduce heat transfer between theraised portions and the recesses. In an exemplary embodiment, a coolingdevice 66, such as cooling or radiator fins, a moving air flow, or acold finger, may also be connected to the sealing device 60. The coolingdevice 66 is connected directly to each of the recessed portions 64 sothat the chambers defined when opposed recessed portions move togetherare maintained at a low temperature. Blood or plasma samples containedin pouches formed within the chamber during the seal process are thusnot damaged by the high temperatures of the heat seal.

[0065] The narrow area (47 of FIG. 3b) through approximately the centerof the seal is formed by an elongated ridge structure 67 provided downthe center of the extended seal head portion 64 of the seal platens. Asthe tubing segment is squeezed between the upper and lower sealingheads, the ridge 67 forces an indentation on the top and bottom surfaceof the seal portion. The indentations narrow the plastic materialcomprising the center the seal, thus making it easy to separate.

[0066] In one embodiment of the invention, the ridge 67 may be serratedin order to provide perforations disposed in a direction orthogonal tothe major axis of the tubing segments. The perforations allow theindividual and connected pouches to be removed from one another withoutthe danger inherent with cutting with a sharp object of violating theintegrity of a pouch by inadvertently cutting through to the samplecontaining area. The perforations are preferably provided during theseal process by providing the seal heads with serrations. Alternatively,perforations may be provided shortly thereafter by use of a separateperforating jig or die.

[0067] Means 68 are also provided to open and close the sealing device60 in order to compress the seal platens together and thus form sealsalong the length of the tubing segment. Such means are well known in theart and may suitably comprise a manual apparatus which opens and closes,such as a lever handle attached to one support frame and which moves theframe against, for example, a hinge. Other suitable arrangements mayinclude vertical guides, springs, or hydraulically operated pistonpresses, or other common mechanical, electrical, or hydraulic presses.

[0068] Turning now to FIG. 4a, there is depicted in semi-schematic view,a specific embodiment of a sealing device 70, useful for providingthermo-compression heat seals at uniform, spaced-apart intervals, so asto form pouches of specific desired sizes, or to isolate linked-togetherY-sites into individual sample-containing aliquots. The sealing device70 suitably comprises top and bottom platens 71 and 72, respectively,adapted to be mounted along the pressure lever and seal band,respectively, of a commercially available impulse sealer, such as one ofthe ALINE M-series impulse sealers, manufactured and sold by the ALINECompany of Santa Fe Springs, Calif. The specific embodiment depicted inFIG. 4a is a two-part heat sealing head adapted to be attached to anALINE MC-15 Impulse heat sealer as an after market modification, andallows the MC-15 to produce pre-filled pouches of plasma for furtherprocessing in accordance with the system and method of the presentinvention.

[0069] The bottom platen 72 of the heat sealing head 70 is constructedof a suitable rigid, heat resistant material such as laminated Kevlar®manufactured and sold by the DuPont Corporation. In the illustratedembodiment, the bottom platen 72 is preferably about 15 inches in lengthin order to fit on the mounting surface of the MC-15 Impulse heatsealer. The bottom platen 72 includes a longitudinal slot 73 which iscentrally disposed and runs along the entire length of the bottom platen72. The width of the longitudinal slot 73 is approximately 0.2 inches inorder to accommodate standard medical tubing, which typically has anouter diameter of approximately 0.1875 ({fraction (3/16)}) inches, innested fashion along the length of the slot.

[0070] A plurality of transverse slots 74 are provided at spaced-apartintervals along the length of the bottom platen 72 which are disposed ina direction orthogonal to that of the central slot 73. The transverseslots 74 have a width of approximately 0.5 inches and are located on1.125 (1⅛) inch centers. Each transverse slot is, therefore, separatedfrom its neighbors by a residual block of platen material centrallydivided by the central longitudinal slot 73 which is about 0.625 (⅝)inches in width.

[0071] Both the longitudinal and transverse slots 73 and 74,respectively, are cut only partially through the material of the bottomplaten 72, thereby forming a substantially flat bed 75 which defines thebottom surface of both the longitudinal and transverse slots. When theapparatus is used to form heat seals, a length of 0.1875 ({fraction(3/16)}) standard medical tubing is nested in position along thelongitudinal slot 73 and rests on the bed 75 of the bottom platen whichfunctions as a bearing surface during the heat seal process.

[0072] A heating element 76, such as a nickel-chromium (NiCr) resistivewire, is provided in a snake-fashion from slot to slot and is disposedlengthwise along each transverse slot comprising the bottom platen inabout the center of the slot. Where the heating element 76 traverses thecenter of the transverse slots 74, the NiCr wire is protected fromcontacting the thermo-sensitive plastic tubing by covering the wire witha piece of, for example, Teflon® tape. Blood or plasma samples containedin the pouches formed within the sealing device during the seal processare thus not damaged by the high temperatures of the heat seal.

[0073] The top platen 71 is also approximately 15 inches in length andis suspended over the bottom platen 72 by the pressure lever of theMC-15 heat sealer. The top platen 71 is constructed from aheat-resistant plastic material such as Lexan® or milled Kevlar® andcomprises a set of equally spaced-apart, generally rectangular teethprotruding from its bottom surface, and extending in a direction towardthe bottom platen. The teeth 77 are about 0.5 inches in length and arespaced-apart on 1.125 (1⅛) inch centers. Accordingly, it can be seenthat each of the teeth 77 is dimensioned to fit into the cavity definedby the transverse slots 74 of the bottom platen 72. Each of the teeth 72of the top platen 71 is positioned to be suspended over a correspondingintersection of a transverse sot 74 and the longitudinal slot 73 of thebottom platen 72. Thus, each tooth 77 is configured to fit into thecavity thus defined when the heat seal platens are closed together byremoval operation of the MC-15 device.

[0074] After a flexible tubing segment is placed within the longitudinalslot 73, the top platen 71 is pushed into contact with the bottom platen72, by lowering the lid of the MC-15 heat seal apparatus. As the lid islowered, the teeth 77 of the top platen 71 enter the cavity defined bythe transverse slots 74 of the bottom platen 72 and contact that portionof the tubing segment which lies exposed on the bed 75 at theintersection of each transverse slot 74 with the central longitudinalslot 73. Current is provided to the nickel-chromium resistive heatingwire which causes the plastic material of the tubing segment to soften.At the same time, the top platen 71 is compressed onto the bottomplaten, thus applying pressure to the plastic material being softened bythe heating element 76.

[0075] After sealing, the tubing segment is labeled on at least one endwith a unique identifier that corresponds to the original plasmadonation. This may be achieved by, for example, gluing a label onto thesegment or by imprinting a bar coded emblem directly onto the tubingmaterial. A prepared recess 78 is suitably provided on the heat sealer70 for holding and aligning a pre-printed bar code identifier tag. Sucha tag is formed from a suitable heat-sealable material and is heatsealed to the tubing segment at the first seal position foridentification purposes. The tubing segment, including the samplecontaining pouches, is then frozen for preservation.

[0076] Returning to FIG. 2, it is important to be able to unambiguouslyidentify all of the various parts of the system that comprise anindividual plasma donation. Thus, unique identifiers such as codedthreads, coded dots, bar codes, or other structure coded with the uniqueidentifier, may be placed in the physical structure of the plasmacollection system For example, in one embodiment, a coded thread 37 ismolded into the donation container 20, a coded thread 39 is molded alongthe edge of the bottle cap 22, a coded thread 41 is molded along theside of the test container 28, and a coded thread 43 is molded into thetubing segments at spaced-apart intervals. The unique identifier in thetubing segment runs along the length of the tubing segments and the codeis repeated in order to permit segmentation of the tubing segments whilemaintaining identification integrity of each segment so prepared.Furthermore, each portion of the donation system is identified with thesame code so that donation identity is maintained for all parts of thesystem.

[0077] Returning now to FIGS. 3a, 3 b, and 4, it may be furtherdesirable to have each individual pouch along a segment identified by analpha or numeric code equal to the position of the pouch along thelinear length of the original tubing segment. Such code may beimprinted, for example, on the compressed portion of the seal padlocated between adjacent pouches by use of a stamping die. Such astamping die may comprise an integral part of the sealing device asdepicted in FIGS. 4 and 4a, so that sealing, forming pouches of variablesizes, and providing narrow or perforated areas for easy separation, aswell as identification numbers, are all accomplished in a singleefficient step. Alternatively, the alpha or numeric identifier couldcomprise part of a perforating jig or die. Stamping dies are known whichinclude means for advancing the alpha or numeric character to a nextsequential one such that sequential pouches in a tubing segment are eachidentified by a corresponding sequential string of alpha (a, b, c .....) or numeric (1, 2, 3, . . . ) characters.

[0078] Therefore, if a first testing pool is being prepared from pouchesfrom several donations, a quality control check may be performed byconfirming that all pouches to be pooled from each tubing segment havethe same location code, for example, number 1. Likewise, when preparinga second testing pool from samples of the same donations, a qualitycontrol check may be performed by confirming that all pouches to bepooled from each tubing segment have, for example, the number 2imprinted at some point on the compressed portion of the pouch.

[0079] In order to effect efficient PCR testing of a donation, theserology test sample taken from each individual donation in testcontainer 28 is tested for various known antigens and/or antibodieswhich are designated for specific viruses. If a sample is positive forone or more known antigen or antibody tests, the individual donation andits corresponding tubing segment are excluded from further testing andboth may be disposed of in an appropriate manner.

[0080] Tubing segments corresponding to the remaining serology negativedonations are divided into identified groups, each group comprising aselected number of donations. As will be described further below, thenumber of donations per group is determined by the sensitivity of thespecific high-sensitivity tests, such as a PCR test, the anticipatedconcentration of the viral RNA or DNA of interest in the plasma sample,and the anticipated frequency of a PCR positive sample occurring withinthe general donor population. For example, for the detection of thehepatitis C virus, containing the RNA of interest, in a population ofrepeat plasmapheresis donors, it is appropriate to pool samples ofbetween 100 and 700 individual donations. For a population in whichviral contamination occurs more often, smaller pools of between 50 and100 individual donations may be appropriate.

[0081] One embodiment of a process of preparing a PCR testing pool inaccordance with the present invention will now be described inconnection with FIGS. 5 and 6. A sampling plate 80, generally similar inapplication to a titer plate but configured in accordance with practiceof the invention, is provided. The sampling plate 80 is configured tocontain generally hemi-cylindrical sample wells 81 disposed horizontallyon the plate in a generally regular array. A suitable sampling plateused to practice the method of the invention has 64 such sample wellsarranged in a 8×8, row/column, rectangular fashion. A cover plate 82having approximately the same exterior dimensions as the sampling plate80 is also provided. The cover plate 82 is adapted to cover the surfaceof the sampling plate 80 in close-fit attachment. Through-holes 83 arearranged on the cover plate in the same array fashion as the samplewells of the sampling plate 80. When the cover 82 is placed over thesurface of the sampling plate 80, through-holes 83 line up verticallyover the sample wells 81, thereby allowing communication with the samplewells through the through-holes. The diameter of the through-holes issubstantially smaller than the surface area of the test sample pouchesand the corresponding sample wells. However, the through-hole diameteris sufficiently large to permit a needle or other cannula like object topass through the holes and enter the sample wells beneath.

[0082] As shown in connection with FIG. 6, a terminal (first generation,“number 1”) pouch 84 is removed from each tubing segment that has beenidentified as belonging to a particular PCR group to be tested. Eachterminal pouch 84 is washed, but not opened, and placed in acorresponding sample well 81 of the sampling plate 80. The cover plate82 is secured over the top of the sampling plate 80 and the plate,cover, and pouches are thawed at an appropriate temperature.

[0083] An equal volume of between about 0.02 to 0.5 ml of plasma isremoved from each pouch and pooled in a testing container. A needle 85or other cannula like device is inserted through the through-hole in thecover plate and into the sampling plate sample well directly below,thereby piercing the tubing material of the side wall of the pouch andgaining access to the plasma sample therein. In an exemplary embodiment,the needle is connected to a device that provides a continuous vacuum orsuction to extract all of the blood or plasma contained in the pouch andminimize any leakage of fluid into the surrounding tray. The needle maybe held in a device which allows the needle to move through thethrough-hole and top wall of the pouch, but restricts its downwardprogress so that the needle is prevented from touching or piercing thebottom wall of the pouch as the pouch sits in the sample well. When thecannula is withdrawn after extracting a sample, the cover plate material86 surrounding the through-hole prevents accidental withdrawal of thepouch along with the cannula, as depicted in FIG. 6.

[0084] While the method of preparing a PCR test pool has been describedin terms of manually extracting a sample by inserting a cannulaindividually into each sample well, the method may equally be practicedusing an automated process. The sampling plate containing pouches ineach well may be held so as to allow an array of cannulas, arranged in amanner corresponding to the arrangement of through-holes in the coverplate, to be pressed down onto the sampling plate, thereby allowing allof the sample pouches to be pierced and samples extracted therefrom atthe same time. Alternatively, a single cannula or cannula holding devicemay be automated or programmed to successively pierce and withdraw fluidfrom each pouch. In order to prevent carryover contamination, a cleancannula is used to withdraw samples for each pool.

[0085] In addition, it will be evident to one having skill in the artthat the combination of sampling plate, sample wells, cover,through-holes, and cannula, while described in connection withextracting sample fluid from a sample packet, is equally applicable toextracting sample fluid from the Y-site sample containers of FIG. 2a.The configuration of the sample wells of FIGS. 5 and 6 are determined bythe shape of the fluid-holding container, and only minor modificationsare required to reconfigure them for Y-sites. For example, the samplewells may comprise an elongated cylinder, oriented vertically, intowhich each Y-site is inserted. A notch may be provided at someappropriate location about the upper periphery of each sample well whichfunctions as a detent into which the Y-site's branch port may bepositioned. This would also function to orient each Y-site and provideadditional positional security. In the same manner as described inconnection with FIGS. 5 and 6, fluid may be extracted from each Y-siteby inserting a cannula through each Y-site's access port and into fluidcommunication with the sample. As the cannula is removed from the accessport, the cover plate material surrounding each through hole acts as astop and prevents the Y-site from being withdrawn from the sample well.

[0086] It will be further evident to one having skill in the art thatthis configuration is equally suitable for practice of the inventionusing an automated process. An array of cannulas may be arranged in amanner corresponding to the arrangement of through-holes in the coverplate, thereby allowing all of the access ports of the Y-sites to bepierced and samples extracted therefrom at the same time. Alternatively,a single cannula or cannula holding device may be automated orprogrammed to successively pierce each access port and withdraw fluidfrom each Y-site.

[0087] An additional embodiment of an apparatus and method suitable forpreparing a PCR testing pool in accordance with the present inventionwill now be described in connection with FIGS. 7, 8, 9 a, 9 b, and 10.Turning first to FIG. 7, a plasma donation pool comprising expressedfluids from a multiplicity of plasma samples is prepared from a numberof plasma donation sample packets in an electrically powered hydraulicpress 90. The hydraulic press 90 suitably comprises a crushing cylinder91 in which sample packets are placed, and a hydraulically operatedpiston 92 which crushes the sample packets. The samples contained withinthe packets are expressed from the crushing cylinder 91 by a suitablecompressed gas, such as compressed air or nitrogen, and collected in apooling container as a pool.

[0088] Initially, a generational pouch (for example, pouch # 1) isremoved from each tubing segment that has been identified as belongingto a particular PCR group to be tested. Each generational pouch iswashed, but not opened, and placed within the crushing cylinder 91 ofthe press 90. Loading of the crushing cylinder is performed within theenvironment of a class II biosafety hood and air-flow path so as toensure against inadvertent contamination of the surrounding environmentby a packet which has lost structural integrity. In a manner to bedescribed in greater detail below, the crushing piston 92 is firmlyseated into the open throat 91 a of the crushing cylinder 91 in such amanner that containment of the contents of the crushing cylinder 91 isassured and that the cylinder 91 and piston 92 combination completelyencloses the sample packets. The manner in which the crushing piston 92engages the crushing cylinder 91 is designed to ensure that theenvironment outside of the cylinder 91 is protected from contaminationby any harmful viruses that may be present in any of the samplescontained by the sample packets.

[0089] The crushing cylinder 91 is next mounted on a cylinder seat 93which aligns the cylinder in correct position on the hydraulic press 90and further allows a hydraulic shaft 94, operatively connected to ahydraulic cylinder 95, to align with and mate to the crushing piston 92.In a manner that will be described in greater detail below, the crushingpiston 92 is releasably connected to the hydraulic shaft 94, such thatthe piston 92 can be both raised and lowered by operation of thehydraulic cylinder 95.

[0090] After the cylinder 91 and piston 92 have been properly aligned onthe cylinder seat 93 and connected to the hydraulic cylinder 95 throughthe shaft 94, a control valve 96 is operated so as to cause thehydraulic cylinder to exert a force on the shaft 94 and piston 92 which,in turn, crushes the sample packets within the crushing cylinder 91. Thehydraulic cylinder 95 operates in conjunction with a four horsepower 240volt AC electric motor 97 which operates a hydraulic reciprocating pump98 which pumps hydraulic fluid in conjunction with a fluid reservoir 99to thereby operate the cylinder 95. About 4,000 lbs of force is pointloaded at the hydraulic shaft 94 which develops a pressure of about 800to 900 psi applied to the sample packets by the piston 92.

[0091] After the sample packets have been crushed, the fluid donationsamples contained therein are expressed from the crushing cylinder 91 bya compressed gas supplied by, for example, a compressed air cylinder 100which is connected through a pressure regulator 101 to a pop-off valve102 provided in the crushing piston 92. In order to allow the pop-offvalve 102 to operate correctly, the piston 92 is first raised slightlyfrom its fully extended crushing position. Compressed air is vented intothe cylinder 91 through the pressure regulator 101 until the thresholdpressure of the pop-off valve is reached. The valve 102 then opens,allowing the compressed gas to pressurize the interior of the cylinderwhich forces the plasma pool out of the crushing cylinder 91 through acollection port 103 provided in the bottom of the cylinder. The plasmapool is then collected in a pooling container connected to thecollection port 103 by an express line or tube as the fluid is forcedout of the cylinder by the compressed air. The compressed air isexhausted into a class II biological safety hood, after passage througha bleach trap.

[0092] Turning now to FIG. 8, there is depicted a cross-sectional,partially cut-away view of a crushing cylinder 91 constructed inaccordance with principles of the invention. The crushing cylinder 91suitably comprises a generally circular base plate 105 having a top andbottom surface and a circumferential lip 106 extending in an upwardlydirection from the top surface, with threads cut into its interior face.A cylindrical cylinder wall 107, open at both ends, is threaded on theexterior face of its bottom end. A rabbet or notch 108 is cut into theinterior face of the bottom end of the cylinder wall 107 so as to definean annular lip 109 which is disposed parallel to the top surface of thebase plate 105 and presents an opposing face thereto. As the cylinderwall 107 is screwed into the base plate 105, a screen plate 110 disposedon the surface of the base plate 105 is engaged by the annular lip 109of the cylinder wall 107 and compressed between the annular lip 109 andthe top surface of the base plate.

[0093] Turning now to FIGS. 9a and 9 b, the screen plate 110 is agenerally circular, disc-shaped plate against which the samplecontaining packets are forced when they are crushed by the crushingpiston 92. As is depicted in FIG. 9b, the screen plate 110 includesfluid gutters comprising radial slots 111 and concentric circular slots112, all approximately {fraction (1/32)} inches in width, which are cutin the top surface of the screen plate. The radial slots 111 are cut atan angle which slopes toward the center of the screen plate 110 wherethey terminate into an axially located drain or sump 113 which drainsthrough a ¼ inch drain pipe 114 (best seen in FIG. 8) drilled throughthe base plate 105.

[0094] Returning now to FIG. 8, a seal is formed between the cylinderwall 107 and the screen plate 110 by engaging and compressing an O-ring115, provided in a seal race 116 cut into the base plate 105 for suchpurpose. The seal race 116 is located in the base plate such that theO-ring 115 lies beneath the vertical intersection of the screen plate110 and the cylinder wall 107. A step 117 is cut into the base plate105, and a mating groove 118 is cut into the screen plate, so that apositive detent is able to precisely locate the screen plate onto thebase plate for proper alignment with the O-ring such that the cylinderwall 107 will properly engage the screen plate and their intersectionwill properly engage the O-ring 115.

[0095] Turning now to FIG. 10, there is depicted, in cross-sectionalpartial cut-away view a crushing piston 92 provided in accordance withprinciples of the invention. The crushing piston 92 comprises agenerally cylindrical piston head 120, having an axially extending,centrally located cup 121 protruding therefrom, the cup 121 havinggenerally cylindrical walls and one open end to define thereby a socket123 for receiving a generally cylindrical hydraulic cylinder shaft 94.

[0096] An annular flange 122 is provided around the circumference of thecylindrical cup 121, and surrounds the cup's open mouth. The outersurface of the flange 122 is beveled, such that the beveled surfaceincreases in diameter in the direction towards the bulk body of thepiston head 120. As the hydraulic shaft 94 is advanced into the socket123, a pair of spring-loaded retaining clips 124 are advanced over thebeveled surface of the annular flange 122 until they detent intoposition and grip the underside of the annular flange.

[0097] To accommodate mating with the socket 123, each retaining clip124 includes a beveled tooth 125 which rides along the beveled surfaceof the piston head's annular retaining ring 122, thereby spreading openthe jaws of the spring loaded retaining clips 124. As the hydraulicshaft 94 continues to advance, the beveled teeth 125 of the retainingclips 124 are eventually advanced past the beveled surface of theannular retaining ring 122. The spring loading of the retaining clipsforces the beveled teeth into contact with the outer surface of the cupside wall. The teeth of the retaining clips 124 are thus engaged withthe underside surface of the annular retaining collar 122, therebygripping the crushing piston 92 and providing means for causing thepiston to move in both directions.

[0098] In addition, it will be evident to one having skill in the artthat the spring loaded retaining clips 124 may be easily disengaged fromthe annular retaining ring 122 by a simple squeezing together of theends of the clips opposite the beveled retaining teeth 125. Accordingly,it will be seen that the piston head 120, the cylindrical, axiallymounted cup 121, the annular retaining ring 122 and the retaining clips124, in combination, provide means for quickly and easily disconnectingthe hydraulic shaft 94 from the crushing piston 92. Thisquick-disconnect feature allows the piston 92 and cylinder 91combination to be easily removed from the cylinder seat 93 of thehydraulic pressure 110 for cleaning, sterilization, refilling withadditional sample packets, and the like.

[0099] As is shown in FIG. 10, the crushing piston 92 further includesseveral O-rings 126 disposed in seal races 178 provided about theperiphery of the piston head 120. The O-rings are provided in order toform a tight pressure seal between the exterior circumferential surfaceof the piston head 120 and the inner circumferential surface of thecylinder wall 107 of the crushing cylinder 91. Multiple O-ring sealsprovide a measure of safety and security, in order to ensure containmentof potentially contaminated sample fluid within the confines of thecylinder 91. While three O-rings 126 are depicted in the illustratedembodiment of FIG. 10, it will be evident that a greater or lessernumber of O-ring seals may be provided in accordance with the invention.All that is required is that a seal be formed between the crushingpiston 92 and the crushing cylinder 91 so as to ensure containment ofpotentially contaminated fluid within the cylinder.

[0100] Returning now to FIG. 8, the crushing cylinder side wall includesa 0.020 inch beveled step 130 which is machined into the interiorsurface of the side wall. The first approximately 1.0 inches, from thetop, of the cylinder side wall 107 is thus, machined to have an insidediameter (ID) approximately 0.040 inches larger than the ID of theremaining portion of the cylinder side wall 107 which extends downwardlytowards the screen plate 110 and base 105. The interface between thestep and the remaining side wall portion is beveled, so as to provide arelatively smooth, angled transition from the slightly larger upper ID,to the slightly smaller lower ID.

[0101] The step on the cylinder side wall 107 is provided so that thecrushing piston 92 may be manually inserted into the open throat of thecrushing cylinder 91 with only slight contact being made between theO-rings (126 of FIG. 10) and the ID surface of the cylinder. Once themanually assembled piston and cylinder combination is placed on thecylinder seat (93 of FIG. 7) the hydraulic shaft 94 is advanced to matewith the socket 123 of the piston and is extended until the retainingclips 124 detent against the underside surface of the piston head'sannular retaining collar 122. The hydraulic shaft 94 is then furtheradvanced so as to push the piston further into the cylinder, therebypushing the O-rings beyond the step 130 on the ID of the cylinder wall.When pushed beyond the step, the O-rings fully compress between the IDof the cylinder side wall 107 and the piston seal races 127, formingthereby a tight seal.

[0102] In operation, the crushing piston 92 develops pressure of about800 to 900 psi (4,000 lbs of force point loaded at the hydraulic shaft)which is a sufficient pressure to crush the sample packets containedwithin the cylinder. Blood or plasma sample fluid flows along the fluidgutter provided in the screen plate and into the central sump, where itis collected and allowed to flow out the extraction port and into apooling container. Following the crushing operation, the hydrauliccylinder 95 is operated to raise the crushing piston 92 a small distance(approximately ½ to 1 inches) above the mass of crushed sample packets,thereby creating a chamber within the cylinder. A pressurized gas, suchas compressed air, is forced into the chamber through the pop-off valve102 in the piston 92. Pressurizing the chamber causes any remainingblood or plasma sample fluid to be expressed out of the cylinder throughthe outlet port 103 into the pooling container.

[0103] Once the crushing and pooling operation is completed, the expressline connected to the outlet port 103, is clamped, to prevent anyadditional sample fluid from exiting the cylinder. The express line isplaced into a bleach container, and the hydraulic cylinder 95 is causedto raise the piston further in the cylinder, thereby creating a suctionwhich siphons bleach from the container into the cylinder. Preferably,the crushing and bleach siphoning steps are repeated two additionaltimes, in order to ensure that any blood or plasma sample “flash back”fluid is fully expressed from the crushing cylinder 91 and that thebleach has ample opportunity to fill the interior volume of the crushingchamber, thereby reducing any gross viral contamination that may befound within.

[0104] Next, the quick release clamps are operated and thepiston/cylinder combination is removed from the hydraulic press 90 andsubjected to sterilization procedures in, for example, an autoclave. Thepiston and cylinder may be subsequently chemically cleaned by soakingthem in a 10% bleach solution for fifteen minutes, followed by a rinsecycle of H₂O, 1% SDS (sodium dodecyl sulfate) surfactant, and H₂O again,prior to autoclaving. If there is insufficient time for autoclavesterilization, the chemical clean may be concluded with a 70% ETOH andsterile H₂O solution. If such additional chemical cleaning is desired,it is performed in a class II biosafety hood which exhausts through aHEPA filter. While under the hood, the crushing cylinder is loaded witha next group of sample packets to be crushed and the crushing piston 92is manually inserted into the open mouth of the crushing cylinder 91 andforced down until the piston's O-rings make contact with the beveledstep formed in the side wall of the cylinder. The newly reloadedcylinder/piston combination is now ready to be placed on the cylinderseat 93 of the crusher 90. The hydraulic cylinder 95 is operated tocause the hydraulic shaft 94 to lower onto the piston 92 such that thequick release clamps engage the annular retaining ring on the piston.The crushing, expressing, and bleach-cleaning process is now repeated.

[0105] From the foregoing, it will be evident to one having skill in theart that the electrically operated hydraulic press (the crusher) 90allows harvesting of blood or plasma samples from a great number ofsample packets in a minimal amount of time. The number of sample packetsable to be crushed by such an apparatus is limited primarily by thescale of the device and the pressure able to be developed by thecrushing piston against the mass of sample packets contained in thecylinder. The 800 to 900 psi of pressure developed by the hydraulicpress of the illustrated embodiment is sufficient to completely crush upto 64 sample containing packets of the type described in connection withFIG. 2. Accordingly, large scale pools comprising up to 512 samples, canbe formed by 8 operation cycles of the crusher of the present invention.This would provide a significant reduction in pool formation time over amethod in which 512 sample packets were individually accessed by acannula to harvest the samples therefrom.

[0106] In addition, it will be apparent to one having skill in the artthat a single large scale pool, comprising up to 512 samples or more,can be formed from a crusher apparatus made sufficiently large enough toaccommodate the greater number of sample pockets in the cylinder. Thehydraulic press portion would also be increased in size to providegreater crushing power to overcome the greater resistance of theincreased number of pockets. As was mentioned above, the pool size wouldonly be limited by the desired scale of the crusher.

[0107] Referring now to FIG. 11, there is shown a flow chart of a PCRtest methodology according to the invention, which allows for theidentification of a unique PCR positive donation with the fewest numberof individual tests.

[0108] The process begins at block 200 with the definition of anappropriate initial pool size which, in turn, depends on various factorssuch as the frequency of occurrence of the virus of interest in thegeneral donor population, the likely final concentration of viral DNA orRNA after dilution in the pool, and the like.

[0109] Although the PCR test is highly sensitive and is capable ofdetecting a single virus in a contaminated sample, a virus mustnecessarily be present in the sample for the PCR test to provide apositive result. If, for example, a sample from a contaminated donationhaving a relatively low virus concentration is pooled together with alarge number of uncontaminated samples, the concentration of virus inthe resulting pool may be so low that there is a statistical probabilitythat no virus is present in a sample taken from the pool for PCRtesting. Such pools may, indeed, falsely test negative for viralcontamination.

[0110] For example, if a 0.02 ml sample was prepared from a plasmadonation contaminated with viruses at a concentration of 500 viruses perml of sample, the 0.02 ml sample would comprise, on average, 10 viruses.If this 0.02 ml contaminated sample were pooled with approximately 500other 0.02 ml samples from uncontaminated donations, the resulting 10 mlpool would comprises viruses at a concentration of 1 per ml.Accordingly, if a 1 ml sample were taken from the pool for PCR testing,there is a significant statistical probability that the PCR sample willcontain no viruses.

[0111] Such low concentrations of virus contamination pose little threatfor products produced from plasma, because several methods are availablefor inactivating viruses present in such low concentration donations.Such viral inactivation methods include the use of solvent/detergent orheating at over 60° C. for an appropriate time or the like. Thesemethods, generally, are described as being capable of reducing theconcentration of viruses by a number of “log units.” For example, thesolvent detergent method is capable of reducing the viral contaminationof hepatitis C by at least 10⁷ per ml or “7 log units.” Thus, plasmaproducts such as factor VIII, factor IX or prothrombin complex may beprepared from plasma donations routinely treated by, for example, thesolvent detergent method after having been PCR tested negative.

[0112] For blood products, routinely transfused directly to a recipient,there remains some small risk of low concentration viral contamination,after such donations have PCR tested negative.

[0113] In the embodiment illustrated in connection with FIG. 11, thefactors discussed above, such as the frequency of occurrence of thevirus of interest in the donor population and the likely concentrationof the virus after dilution, are evaluated. An appropriately sized firstlevel PCR testing pool is designed which minimizes the statisticalprobability that viruses present in low concentrations will goundetected. The pool is prepared at block 201 by pooling the contents ofterminal pouches of identified tubing sections, in the manner describedabove. At block 202, a PCR test is performed on the first level PCRpool.

[0114] Block 203 represents a decision point in the methodology of theinvention which depends on the results of the PCR test performed inblock 202. In the event of a negative result on the test, all of thedonations corresponding to samples used to make up the first level PCRpool are presumed to be free of viral contamination and released forfurther processing into pharmaceutical products. The methodology thusexits on receipt of a negative PCR test result. When the PCR testreturns a positive indication, this indicates that a viral contaminantis present in one, or more than one, of the donations which made up theoriginal PCR first level pool. At block 204, an additional sample pouch,the pouch next to the one first removed, is taken from tubing segmentswhich correspond to donations comprising the original PCR first levelpool. These additional sample pouches are divided into two approximatelyequal subgroups, designated A and B herein for purposes of clarity.

[0115] These subgroups are then separately pooled using a separate,clean cannula to form each subgroup pool in the same manner as describedabove, and only one of the subgroup pools is PCR tested, as indicated atblock 205. It is immaterial for purposes of the invention which of thetwo subgroups is tested. In block 205, subgroup A is identified as thesubgroup to be tested, but subgroup B could just as easily have beendesignated without disturbing the methodology of the invention.

[0116] At block 206, a decision is made depending on the outcome of thePCR test of subgroup pool A. In the event that subgroup pool A testsnegative for a PCR viral indication, no further testing is performed onsamples from donations that comprised subgroup A. Rather, as indicatedat block 207, the next sample pouches in sequence are taken from tubingsegments that comprised subgroup B which are then, in turn, divided intotwo approximately equal subgroups A′ and B′. Each subgroup in this stepcomprises approximately half the number of samples as comprised theimmediately preceding subgroup. The contents of the subgroup samplepouches are again pooled separately in the same manner as describedabove. In the event that subgroup A tested PCR positive, indicating atleast one of its component donations was virus contaminated, the otheruntested subgroup (subgroup B in the example of FIG. 11) is now PCRtested at block 108 to confirm that it is not also PCR positive.Subgroup A now becomes the subgroup further subdivided into twoapproximately equal subgroups (A′ and B′), as indicated at block 209.

[0117] At block 210, PCR testing is performed on only one of thesubgroup pools, A′ or B′, defined in preceding step 207 or 209. Themethod now iterates and returns to block 206, wherein the decision stepis applied to the results of the PCR test performed at block 210. Again,if the PCR test results prove negative for the tested subgroup, theuntested subgroup would be further subdivided into two approximatelyequal subgroups, each comprising approximately half the samples of thepreceding subgroup. If the tested subgroup returned a PCR positiveresult, the tested subgroup would be further subdivided into twoapproximately equal subgroups, each of which would comprise one half ofthe samples of the preceding: subgroup. In this case, the untestedsubgroup would again be PCR tested in order to confirm that it was notalso PCR positive.

[0118] The test methodology continues iterating from block 206 throughblock 210 until testing is determined to be complete. Test completion isdefined as when a subgroup division results in the creation of twosubgroups, each containing only one sample pouch corresponding to asingle donation. One of the samples is PCR tested at block 210 and, ifthe test results are negative, the other sample is identified asbelonging to a virally contaminated plasma donation. If the testedsample tests positive, the remaining sample is then also PCR tested inorder to confirm that it is not also PCR positive.

[0119] Upon completion of all testing, the methodology of the inventionends at block 211. It should be clear from the flow chart of FIG. 11,that the testing methodology of the invention only requires that two PCRtests be performed at each test level when the initially tested pool ispositive: One initial test for one of the two subgroups, and onesubsequent test to confirm that the corresponding initially untestedpool is indeed negative. The test methodology requires only a single PCRtest at each test level when the initially tested pool is negative.

[0120] Application of the system and method for sample testing of theinvention will now be described in connection with a particular PCR testpool size, as depicted in FIG. 12. In FIG. 12, the terminal pouches of512 individual donations are formed into an initial PCR testing pool at212. For purposes of illustration, it will be assumed that only one ofthe 512 samples was taken from a donation which was contaminated by avirus of interest. The tubing segment depicted in FIG. 12 whichcomprises 10 individual and connected pouches represents the tubingsegments originally connected to and taken from the contaminated plasmadonation container.

[0121] The initial 512 sample pool is PCR tested and because of thepresence of the contaminated sample, returns a positive viralindication. At step 213, two 256 donation pools (256A and 256B) areprepared from the next sequential pouches taken from segments that madeup the prior positive pool. Pool 256B is now PCR tested and, as depictedin FIG. 12, returns a negative viral indication, thus indicating thatpool 256A contains a sample from the contaminated donation.

[0122] At step 214, two 128 donation pools are prepared from the nextsequential pouches of tubing segments that made up pool 256A. Thus,according to the invention, pool 256A has been subdivided without havingbeen PCR tested. At step 203, pool 128A is now PCR tested and, since itreturns a negative viral indication, pool 128B is now known to include asample pouch from the contaminated donation. Pool 128B is thensubdivided into two 64 donation pools (64A and 64B) by removing the nextsequential pouch from those tubing segments whose preceding pouches madeup pool 128B.

[0123] Next, pool 64B is PCR tested and, in the example of FIG. 12,returns a positive viral indication. In this case, PCR testing isperformed on pool 64A in order to verify that it is, indeed, negativeand that no additional contaminated samples are present beyond those inpool 64B. At step 216, pool 64B is further subdivided into two 32donation pools, 32A and 32B, by removing the next sequential pouch fromtubing segments used to make up preceding pool 64B. Pool 32B is PCRtested, returns a negative viral indication, as indicated, and pool 32Ais therefore further subdivided into two 16 donation pools, 16A and 16B.Again, the 16 donation pools are prepared by removing the nextsequential sample pouch from tubing segments that made up the precedingpositive pool, 32A.

[0124] At step 217, pool 16B is PCR tested and returns a positive viralindication. Pool 16A, therefore, is PCR tested in order to confirm thatit is negative, and that all contaminated samples are present in pool16B.

[0125] At 218, pool 16B is subdivided into two 8 donation pools, 8A and8B, by removing the next sequential sample pouch from tubing segmentsthat made up the preceding positive pool 16B. Pool 8B is then PCR testedand, as illustrated, returns a negative viral indication, indicatingthat pool 8A contains a sample from a contaminated donation. Pool 8A isthen further subdivided into two 4 donation pools, 4A and 4B, at step219. PCR testing is performed on pool 4B, which returns a negativeindication, thus indicating that pool 4A contains a sample from acontaminated donation. Pool 4A is then subdivided, at 220, into pools 2Aand 2B in the same manner, as described above. Upon PCR testing, pool 2Areturns a negative viral indication indicating that one of the twosamples comprising group 2B was taken from a tubing segment of acorresponding contaminated donation.

[0126] At step 221, the individual donations are tested by removing thefinal pouch from the tubing segments that made up group 2B. The finalindividual donations are PCR tested in order to identify the specificpositive donation, which is then removed from storage and appropriatelydisposed of. The remaining 511 viral free donations are retained forfurther processing into pharmaceutical products.

[0127] In the above example, a single contaminated donation has beenuniquely identified from a group of 512 such donations, by performingonly 13 separate PCR tests, including the primary PCR test on theoriginal 512 donation pool. The method of the invention, allows forskipping a PCR test on a particular subpool, so long as thecorresponding tested subpool returns a negative viral indication. Bythus skipping certain PCR tests, the method of the invention reduces thenumber of PCR tests that must be performed in order to identify aspecific positive donation, without sacrificing the resolution of thePCR test methodology. Under the method of the invention, all positivedonations will be identified but without requiring that all donations betested.

[0128] From the exemplary embodiment of FIG. 12, it will be clear thateither one of the successively smaller subgroups may be PCR tested andthat the arbitrary position of the positive sample may be varied. Thus,if a sample from the positive donation were present in each initiallytested subpool, 18 tests would be required to uniquely identify thepositive donation (one initial test which returns a positive indicationand one additional test to assure that the corresponding subpool isnegative).

[0129] By the same token, if each initially tested subpool returns anegative indication, 10 tests would be required to identify the positivedonation. In practice, positive and negative test results on thesubpools would tend to distribute equally, thus, 14 tests on averagewould be required to identify a uniquely positive donation from aninitial donation pool for 512 units.

[0130] It is therefore clear from the foregoing that the system andmethod of the present invention, including the provision of tubingsegments comprising individual and connected pouches each containing asample of a plasma donation, is advantageous in providing a multiplicityof PCR test pools. Unlike conventional pool preparation, in which asequence of initial and subsequent pools are formed from a single sampleof each donation at the same time, the present invention allows forformation of a test pool immediately prior to testing. This manner of“just-in-time” pool formation permits construction of test pools fromindividual pouches only as needed. The possibility of contamination iseliminated since the pools are constructed at different times, each fromsealed sample pouches. Moreover, sample pouches remain frozen untilneeded to develop a test pool. Multiple freeze-thaw cycles which mayadversely affect the recovery of the DNA or RNA of interest are avoided,thus insuring the integrity of the PCR test.

[0131] While the above-described method is effective for identifying aviral positive donation with the fewest number of relatively expensivePCR tests, other methods for identifying individual positive donationsare also provided in accordance with practice of the present invention.In particular, one such method has the property of being able toidentify individual positive donations within two to three PCR testingcycles, thus significantly reducing the amount of time andadministrative overhead required to screen a large number of donations.

[0132] For example, in the above-described method, once a particularsubpool has been identified as containing a positive donation, atechnician must identify those donations which contributed samples toform that particular subpool. Those donations must then be revisited,and an additional sample packet must be harvested from eachcorresponding tubing segment. Two next-generation subpools must then beformed, and PCR testing repeated. This harvesting, subgroup poolformation, and PCR testing process is repeated for smaller and smallergenerational subpools until the method uniquely identifies the virallycontaminated donation.

[0133] However, a significant amount of time is consumed in each PCRtesting cycle (harvesting, subgroup pool formation, and PCR testing).Taking the 512 sample first generational pool as an exemplar, it will beevident that at least 10 PCR testing cycles will be required to identifya unique viral contaminated donation. While highly cost-effective, theabove-described method may present challenges to a PCR testinglaboratory when time is of the essence.

[0134] A methodology for uniquely identifying viral positive blood orplasma donations in the fewest number of PCR testing cycles will now bedescribed in connection with FIGS. 13 and 14.

[0135] Turning now to FIG. 13, there is depicted a flow chart of a PCRtesting methodology, in accordance with the invention, for efficientlydetecting a PCR positive individual donation in a pool with the minimumnumber of PCR analysis cycles. As was the case with the prior-describedPCR testing method, the method of FIG. 13 assumes that the PCR test hassufficient sensitivity to detect the presence of a positive sample in apool of the appropriate size. For purposes of illustration only, theinitial grouping has been chosen to represent 512 blood or plasmadonations. It will be understood by those having ordinary skill in theart that the initial grouping size may be larger or smaller depending onthe particular genome marker being evaluated, the sensitivity of the PCRtest procedure used, the expectation value of the genome markerconcentration within a sample aliquot, and the sample aliquot size.

[0136] The method begins in block 301 by defining an N-dimensionalsample matrix or grid. The matrix may be of any size and comprise anynumber of dimensions from 2 to N, but preferably is a 3-dimensionalregular matrix, organized as a square.

[0137] An example of such a matrix is depicted in FIG. 14, which is agraphical illustration of square matrix, characterized by 3-dimensional;row, column, and slice (r,c,s). In the exemplary matrix of FIG. 14,there are 3 rows, 3 columns, and 3 slices, thereby defining 3³, or 27,elements. In the exemplary embodiment, a row is considered as comprisingall of the elements defined by taking an imaginary vertical sectionthrough the square regular matrix. In the embodiment of FIG. 14, theelements comprising, for example, row 3 of the matrix are identified bythe letter r₃ on their row faces.

[0138] Likewise, a column comprises all of the matrix elements definedby taking a second imaginary vertical section through the matrix, in adirection orthogonal to the direction of a row. In the exemplaryembodiment of FIG. 14, the elements that comprise, for example, column 1have the letter c₁ on their column faces. A slice is defined as allelements comprising a horizontal section taken through the exemplarymatrix of FIG. 14. In like manner to the row, column, definition, theelements comprising slice 1 are identified with the letter s₁ on theirslice faces.

[0139] It can be seen, therefore, that each of the 27 elements in thematrix of FIG. 14 uniquely belongs to 1 of the 3 rows, 1 of the 3columns, and 1 of the 3 slices. Mathematically, this may be expressed bythe relationship X_(rcs), where X denotes an element, and rcs is adimensional index, where each of the indices may take on a value from 1to 3. The specific element X₁₁₃ may be identified as that element at theintersection of row 1, column 1, and slice 3.

[0140] From the foregoing, it will be apparent that although theexemplary matrix of FIG. 14 is a 3×3×3 matrix, the principles of matrixdefinition and element formation will hold for matrices with a muchgreater number of rows, columns, and slices. In particular, an 8 row, 8column, 8 slice matrix may still be represented mathematically asX_(rcs), where rc and s may now take on values from 1 to 8. Thus, a3-dimensional 8×8×8 matrix is able to accommodate identifiers for 512elements.

[0141] Returning now to the method flow diagram of FIG. 13, followingdefinition of an N-dimensional sample matrix, particular blood or plasmadonation samples are mapped to each of the elements defined by thematrix. In an exemplary 3-dimensional 8×8×8 matrix, a sample from eachof 512 individual donations is associated with a matrix element, andidentified by a corresponding, unique X_(rcs) indicator.

[0142] Next, an aliquot is taken from each sample, and a multiplicity ofminor sub-group pools are formed. Each minor pool comprises the aliquotsof all of the samples (X_(rcs)) in which 1 of the dimensional indices isfixed. In other words, in accordance with the above-described exemplarymatrix, all of the samples (X_(rcs)) which have r=1, regardless of thecolumn or slice value, are formed into a minor pool; likewise for r=2,r=3 . . . r=N; likewise for c=1, regardless of row or slice value, c=2,. . . c=N; likewise for s=1, regardless of row or column value, s=2 . .. s=N. Each minor pool thus represents each row, column, layer, or otherdimensional index, such that if an N-dimensional matrix has beendefined, there will be N-dimensions times the (total number ofsamples)^(1/n) minor pools. For the exemplary 3-dimensional 8×8×8 matrixcontaining 512 samples, there will be 24 minor subgroup pools (8 rowpools, 8 column pools, and 8 layer pools). The creation of minor pools,in accordance with the invention, may be viewed as being similar to themathematical method of reducing a determinant by the method of minors.In like manner, each sample will be understood to be represented in Nminor pools, 1 for each dimension of the matrix.

[0143] In addition to forming the minor pools, an aliquot of eachsample, or an aliquot of each of the minor pools, is combined to form asingle master pool which contains a sample from all of the 512 donationscomprising the present donation space. After all of the pools areformed, any remaining samples and the minor pools and master pool may berefrozen and stored until such time as PCR testing is desired.

[0144] When PCR testing is desired, a PCR test is first performed on themaster pool which represents an aliquot of each sample comprising thematrix. If the test results for the master pool are negative, there are,at least to the sensitivity level of the PCR test, no viral positivedonations represented by samples forming the matrix. The blood or plasmadonations which have contributed samples to the matrix may be releasedfor further use. However, if the PCR test of the master pool is positivefor a particular genome marker, a second PCR testing cycle is entered,at 300, in which each of the minor pools are now tested.

[0145] In a manner similar to that described above, the major pool sizeschosen such that the statistical probability of their being more thanone positive sample in the major pool (the 512 samples) is small,preferable less than 1 to 2%. This can be done by evaluating thefrequency of occurrence of the virus of interest in the general donorpopulation to a 98% to 99% confidence level. For example, if it isdetermined that only 1 donor out of a general donor population of 1,000is contaminated with the virus of interest to a 98% confidence level,there is a 2% probability of finding more than 1 contaminated donor inthe next 1,000 donors being evaluated. This assures that the algorithmwill, in general, be able to identify the single reactive unit in a poolof appropriate size within the PCR testing cycle. In accordance with theinvention, given a single positive sample within the matrix, 3 of theminor pools will contain an aliquot of the positive donation, 1 in eachdimension. In the exemplary embodiment (the matrix of 512 samples),there are 8 minor row pools, 8 minor column pools, and 8 minor layerpools. If the master pool tests positive, then 1 row, 1 column, and 1layer pool will test positive during the second PCR testing cycle asshown at 307. The intersection of the row, column, and layer elementindex unambiguously identifies the reactive donation as shown at 309.

[0146] As an example, if the reactive sample has been mapped to matrixelement X₁₁₃, the row 1 minor pool will return a positive PCR testresult, while the row 2, and subsequent row minor pools will testnegative. Further, the column 1 minor pool will return a positive testresult, while the column 2 and subsequent column pools will testnegative. Likewise, the layer 1 and 2 minor pools will return a negativeresult, the layer 3 pool will test positive, and subsequent layer minorpools will test negative. The 3 positive minor pools (row 1, column 1,and layer 3) have only a single element in common, _(X113). Thus, thepositive donation is uniquely identified as represented by the samplemapped to element X₁₁₃.

[0147] If there is more than 1 reactive donation in the matrix, thereactive donations may still be unambiguously identified by the methodof the invention, by no more than 1 additional PCR testing cycle. If itis observed that more than 1 minor pool of a single dimensional indexreturns a positive test result, while only a single minor poolrepresenting each of the remaining dimensional indices returns apositive test result, the more than 1 positive donations may beunambiguously identified by mathematically evaluating the test resultswithout the need for a third PCR testing cycle.

[0148] For example, if a row 1 minor pool, and none other, testspositive; a column 1 minor pool, and no other, tests positive; and alayer 1 minor pool and layer 3 minor pool both test positive, there areonly two positive donations comprising the matrix, and they are able tobe unambiguously identified as X₁₁₁ and X₁₁₃. No further testing isrequired to arrive at this result.

[0149] If, on the other hand, it is observed that multiple minor poolstest positive and their identities indicate changes along 2 dimensionalindices as shown at 310, it will be apparent that there will be z²elements identified as potentially mapped to a positive donation, wherez is the actual number of positive donations comprising the grid.

[0150] For example, if the row 1 minor pool, and no other, testspositive; the column 1 and column 3 minor pools test positive; and thelayer 1 and layer 3 minor pools test positive, this suggests that thepotentially positive candidate elements are X₁₁₁, X₁₁₃, X₁₃₁, and X₁₃₃.Since there is a multiple in only two of the dimensional indices (columnand layer), and for candidate elements, it will be seen that there areonly two actual positive donations comprising the matrix. In thiscircumstance, all 4 donations may be arbitrarily identified as beingpositive, and disposed of or, alternatively, an aliquot may be takenfrom each of the 4 candidate elements and individually PCR tested duringa third PCR testing cycle at 311, in order to uniquely identify which 2of the 4 comprise the actual positive donations.

[0151] In like manner, it is mathematically evident that if there aremore than 2 positive donations in the matrix, and their identifiers varyin more than 2 dimensions, there will be, at most, z^(n) potentiallypositive candidate elements identified, where z is the actual number ofpositive donations, and where n is the number of dimensions which vary.In this circumstance, aliquots are taken of all suspect elements in thematrix and directly tested.

[0152] Thus, it can be seen that the method of the invention permitsunambiguous identification of donations which are reactive for aparticular genome marker within a single PCR testing cycle for aninitially positive master pool, within 2 PCR testing cycles for allmatrices which contain a single reactive donation or a multiplicity ofreactive donations which vary along only a single dimensional index, andwithin 3 PCR testing cycles for any other situation.

[0153] Accordingly, the practice of the present invention results in theblood supply, and blood or plasma products prepared therefrom, beingsubstantially safer by virtue of its being as free as possible fromviral contamination. Advantageously, cost-effective, high-sensitivitytesting is readily performed for the presence of a virus directly. Thus,false indications of virus contamination usually associated withantibody testing during the infectivity window period is avoided.Moreover, the present invention allows cost-effective use ofhigh-sensitivity tests which are capable of detecting the presence of asingle virus in the test sample, thus helping to insure the freedom ofthe blood supply from incipient viral contamination.

[0154] Those skilled in the art will appreciate that the foregoingexamples and descriptions of various preferred embodiments of thepresent invention are merely illustrative of the invention as a whole,and that variations in the shape, size, and number of the variouscomponents of the present invention, as well as the types of testsimplemented, may be made within the spirit and scope of the invention.For example, it will be clear to one skilled in the art that the lengthof the individual and connected pouches, and therefore their volumetriccontent, may be progressively increased along the length of the tubingsegment. As successive testing subpools are formed from a smaller andsmaller number of samples, the volume of plasma comprising the poolnecessarily decreases. It should be clear that in order to maintain asufficient volume of plasma in each successive subpool, successivesample pouches may contain a larger volume in order to accommodate adesired final pool volume. In order to accommodate pools ranging in sizefrom about 1 ml to about 10 ml, it will be clear that the volumes ofsuccessive sample pouches will increase from about 0.02 ml to about 0.5ml, in progressive steps. In one exemplary embodiment, the pouch volumeis 0.02 ml in the first pouch to be used in the largest pool and is 0.2ml in the final pouch.

[0155] It will also be clear to those skilled in the art that the systemof the invention is not limited to the exemplary plasma collectioncontainer and an associated tubing segment. Blood bags or otherbiological fluid containers may be used with equal facility and suitabletubing segments may be attached thereto both prior to fluid collectionand after fluid collection is completed. All that is required is thatsample quantities of biological fluids be transferred to a tubingsegment which is then formed into pouches in accordance with practice ofthe invention.

[0156] Accordingly, the present invention is not limited to the specificembodiments described herein but, rather, is defined by the scope of theappended claims.

What is claimed is:
 1. A method for forming subpools for use in testingfor presence of a specified agent in a set of fluid samples, the methodcomprising the operations of: (a) associating each of the fluid sampleswith a distinct matrix element of an N-dimensional matrix, each of thematrix elements being identified by values of N indices, the N indicescorresponding one-to-one to the N dimensions of the matrix; (b) formingN aliquots from each of the fluid samples; and (c) combining aliquots toform the subpools such that each of the subpools comprises one aliquotfrom each of the fluid samples associated with matrix elements that havean identical value for one of the N indices.
 2. The method of claim 1,further comprising the operations of: (d) testing each of the subpoolsto detect presence of the specified agent; and (e) producing a positiveindication for one of the subpools when the presence of the specifiedagent is detected in said subpool.
 3. The method of claim 2, furthercomprising the operations of: (f) combining identities of all subpoolsthat have a positive indication to identify at least one correspondingpositively indicated fluid sample.
 4. The method of claim 1, wherein theN indices have an identical range of values.
 5. The method of claim 1,wherein N is equal to
 3. 6. The method of claim 2, wherein operation (d)is a high-sensitivity test.
 7. The method of claim 6, wherein thehigh-sensitivity test is a polymerase chain reaction (PCR) test.
 8. Themethod of claim 3, wherein N is equal to 3 and there are 3 sets of saidsubpools corresponding to the 3 indices, each subpool of the 3 sets ofsubpools being identified by a specific value of a corresponding one ofthe N indices.
 9. The method of claim 8, wherein operation (f) resultsin identifying K positively indicated fluid samples, K being an integergreater or equal to 1, when one of the N sets of subpools has K subpoolshaving positive indication and each of the remaining N-1 sets ofsubpools has only one subpool having positive indication.
 10. The methodof claim 8, wherein operation (f) results in identifying a set ofcandidates for positively indicated fluid samples when more than one ofthe N sets of subpools has more than one subpool having positiveindication.
 11. The method of claim 10, further comprising the operationof: forming a new aliquot from each of the candidates for positivelyindicated fluid samples; testing each of the new aliquots to detectpresence of the specified agent; and producing a positive indication forone of the new aliquots when presence of the specified agent is detectedin said new aliquot, said new aliquot identifying a correspondingpositively indicated fluid sample.